The p53 gene family although small in number, plays important roles in cells and whole animals. p53 itself is a major tumor suppressor that integrates multiple sources of stress with cellular response including arrest, senescence and apoptosis. The p53 homologues p63 and p73 are critical for development based on the phenotype of mice lacking either of these genes, and they may also contribute to tumor suppression. The goal of this project is to understand how p53 members are regulated by previously identified or newly discovered cellular factors and how they interact with each other and with DNA. Aim 1 of this project will focus on the checkpoint kinases Chk1 and Chk2. It will elucidate the structure and function of Chk2 with respect to novel Chk2 phosphorylation sites, Chk2 substrate specificity and interaction with its substrates such as p53. The finding that both Chk kinases through E2F1 control p73 induction after DNA damage will be pursued to elucidate how E2F stability and activity are targeted by Chk1 and Chk2. Whether and how oncogenes abrogate the ability of Chk2 to affect p53 activity will be determined in collaboration with Scott Lowe. With Carlos Cordon-Cardo the properties of newly identified tumor-related Chk2 mutations will be analyzed. The goal of Aim 2 is to identify and characterize proteins that interact with different isoforms of p63. The possible effect of p63 on newly identified proteins will be tested and conversely the effect of new binding partners on p63 activities will be determined. Aim 3 will consist of experiments examining the interactions of p63 and p73 with mutant p53 and with DNA. First, small molecules that can disrupt the interaction between mutant p53 proteins and p63 or p73 will be identified and the cellular outcome of such compounds will be determined. Second, the optimum DNA binding sequences for p63 and p73 isoforms in vitro and in vivo will be determined, and with Arnold Levine an informatics approach will be taken to identify human genes that may be preferentially regulated by these p53 family members.